Servis Catherine, project leader

Catherine Servis received her PhD from the University of Heriot-Watt in Edinburgh on studies involving the localization, identification and synthesis of the immunodominant determinants of Lactate Dehydrogenase-B4 and IgG2a myeloma protein. After a postdoctoral training at the Basel Institute for Immunology (Hoffmann-La-Roche) she continued her training in protein chemistry at the Friedriech- Miescher Institute (Ciba Geigy –Novartis). In 1990, she joined the Institute of Organic Chemistry of the University of Lausanne where she was involved in the development of strategies for the solid phase synthesis of molecules for biological applications. She joined the Institute of Biochemistry in 1992 and she is heading the Protein and Peptide Chemistry Facility. She is head of the Clinical Tumor Proteome Analysis Facility since May 2005.

Interests: Development of clinical proteomic strategies for the identification and quantification of biomarkers in biofluids and tissue from cancer patients. Development of synthetic tools for biological applications.

DEVELOPMENT OF PROTEOMIC STRATEGIES FOR BIOMARKER DISCOVERY AND SYNTHETIC TOOLS FOR BIOLOGICAL APPLICATIONS

Clinical proteomics

Cancer-specific markers in plasma which may be useful for cancer detection and monitoring include proteins released in smaller amounts from cancer tissues (tissue-specific proteins) or as a result of structural and functional changes in the microenvironment surrounding cancer cells. They may also include mediator or effector molecules of the native or acquired immune response to cancer cells (e.g. auto antibodies to cancer-specific proteins). For these reasons, most cancer markers currently in clinical use are low-abundance proteins with concentrations in the nanogram per milliliter range in plasma.

Proteomics technology promises to be a valuable way to identify proteins and fragments thereof present in the tumor environment that may be used to define new molecules for diagnostic, prognostic and therapeutic purposes. Recently, as many as 1261 proteins believed to be differentially expressed in human cancer patients relative to healthy individuals, have been proposed as candidate plasma biomarkers that could be useful in early cancer detection and monitoring, given sufficiently sensitive and speciﬁc tests are available for their detection. In the last decade, different proteomics platforms based on Mass Spectrometry (MS), have been developed and used for the identification of cancer-associated proteins and peptides in tissue and serum.

Activities

1. The Protein and Peptide Chemistry Facility (PPCF) provides synthetic tools for various biological applications and a variety of analytical and technical services using mass spectrometry to the Lausanne scientific community. The Facility is also involved in research and teaching on different aspects of protein and peptide chemistry.

2. The Clinical Tumor Protein Analysis Facility (CTPAF, prior within the NCCR program) will be part of the Molecular Biomarker Facility (MBF) of the nascent Lausanne Cancer Center (LCC). The goal of this platform is to develop and optimize strategies for the identification and quantification of molecular markers in biofluids and tissue from cancer patients.

Research projects

1. Monitoring proteins relevant to tumor angiogenesis and tumor progression by proteomic analysis of biofluids and tissue from cancer patients

i) Targeted analysis of molecules in biofluids and tissues. In a hypothesis-driven approach we will accurately identify and quantify selected proteins in CSF and serum from patients with multiple glioblastoma or other solid tumors (e.g. colon, breast, head and neck cancers). We will detect molecules that have been previously identified by a single biomarker approach (e.g. VEGF-A, MMP-9) and molecules that have been identified as potential candidates of angiogenesis or tumor progression in gene expression studies (e.g. Cyr61/CCN1; GDF3, GDF-15, EPSTI-1). We will employ a quantitative proteomics approach developed by Aebersold which is based on the selection and chemical synthesis of isotopically labeled reference peptides corresponding to peptides generated from the native protein by tryptic digestion. Since the proteins of interest are glycoproteins, we will use the glycoprotein enrichment method which is developed by the same group and further analysis by LC-ESI MS in SRM/MRM mode in the presence of the reference peptide. This approach has been proven suitable for detecting a panel of candidate biomarkers in clinical samples.

ii) Global protein profiling in biofluids and cancer tissues. Once this first approach has been successfully established, we plan to analyze tumor tissue and biofluids for global protein expression profiles (signatures) specific to the tumor, its propensity to progress (patient survival) and (possibly) its response to therapy. We will study only patients for which clinical follow up and/or patient outcome is known. The proposed strategy involves fractionation of the material to ensure characterization of the different types of components (e.g. small vs large proteins). After immunodepletion of the high abundant proteins, samples will be reduced, alkylated and digested by trypsin and generated peptides separated by 2-D LC and analyzed by MALDI-TOF/TOF. Quantitative differences in the proteins present in tumor tissue or serum/CSF samples will be initially determined through comparison of the peak intensities of their constituent peptides/proteins. Later, when specific peaks have been characterized as interesting, we will quantitate the corresponding proteins by the use of isotopically labeled reference peptides (see above).

The aim of this project is to detect the alteration of the glycosylation pattern in the serum of cancer patients by differential analyses using stable isotope labelling. The method developed includes cleavage of N-linked glycans from glycoproteins in serum using PNGase F, and derivatization of the glycans with 2-aminopyridine (or an isotopically labeled variant). The reductively aminated samples are mixed, chemically desialylated and analyzed on a quadrupole time-of-flight system coupled to a micro-HPLC system and a microfluidic device. The different glycoforms are assigned based on their molecular mass, and their structure is confirmed by their MS/MS patterns. The relative ratio of the signal of one glycoform reflects the abundance of the glycan in the two samples.

We will apply this method to the relative quantification of glycans in the serum of patients with colon, breast cancer and controls. In addition to the analysis of the glycans from the whole serum, glycan patterns will also be analyzed after the depletion of the major high abundant proteins in serum. This will provide information about N-glycans from low abundant proteins which may include membrane surface proteins that play an important role in cancer development.

Within this project, our main focus is the analysis (separation and identification) and quantitation of potential HLA class I-restricted ligands generated by proteasomal degradation of precursor peptides encoded by alternatively expressed exons. The proteolytic activities of other peptidases involved in the antigen processing pathway will also be studied and analyzed.

The main goal of the project is to improve the performance of MS-based proteomics analysis and understanding of tandem mass spectrometry fundamentals. The main tasks are related to the development of novel methods of peptide characterization based on sequence dependent retention time (RT) prediction in combination with production abundance analysis based tandem mass spectrometry (MS/MS) and corresponding basic research leading to the application of the novel and improved approaches for analysis of biologically active compounds (peptides).